Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

Abstract

<ns4:p> <ns4:bold>Background:</ns4:bold> <ns4:italic>Wolbachia</ns4:italic> , a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the <ns4:italic>Anopheles (An.)</ns4:italic> genera, but has recently been found in <ns4:italic>An. gambiae</ns4:italic> s.l. populations. As there are numerous <ns4:italic>Anopheles</ns4:italic> species that have the capacity to transmit malaria, we analysed a range of species to determine <ns4:italic>Wolbachia</ns4:italic> prevalence rates, characterise novel <ns4:italic>Wolbachia</ns4:italic> strains and determine any correlation between the presence of <ns4:italic>Plasmodium</ns4:italic> , <ns4:italic>Wolbachia</ns4:italic> and the competing endosymbiotic bacterium <ns4:italic>Asaia</ns4:italic> . </ns4:p> <ns4:p> <ns4:bold>Methods:</ns4:bold> <ns4:italic>Anopheles</ns4:italic> adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017. Molecular analysis of samples was undertaken using quantitative PCR, Sanger sequencing, <ns4:italic>Wolbachia</ns4:italic> multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial <ns4:italic>16S rRNA</ns4:italic> gene. </ns4:p> <ns4:p> <ns4:bold>Results</ns4:bold> : Novel <ns4:italic>Wolbachia</ns4:italic> strains were discovered in five species: <ns4:italic>An. coluzzii</ns4:italic> , <ns4:italic>An. gambiae</ns4:italic> s.s., <ns4:italic>An. arabiensis</ns4:italic> , <ns4:italic>An. moucheti</ns4:italic> and <ns4:italic>An.</ns4:italic> species ‘A’, increasing the number of <ns4:italic>Anopheles</ns4:italic> species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with <ns4:italic>Wolbachia</ns4:italic> supergroup B strains. We also provide evidence for resident strain variants within <ns4:italic>An</ns4:italic> . species ‘A’. <ns4:italic>Wolbachia</ns4:italic> is the dominant member of the microbiome in <ns4:italic>An. moucheti</ns4:italic> and <ns4:italic>An.</ns4:italic> species ‘A’, but present at lower densities in <ns4:italic>An. coluzzii</ns4:italic> . Interestingly, no evidence of <ns4:italic>Wolbachia/Asaia</ns4:italic> co-infections was seen and <ns4:italic>Asaia</ns4:italic> infection densities were also shown to be variable and location dependent. </ns4:p> <ns4:p> <ns4:bold>Conclusions:</ns4:bold> The important discovery of novel <ns4:italic>Wolbachia</ns4:italic> strains in <ns4:italic>Anopheles</ns4:italic> provides greater insight into the prevalence of resident <ns4:italic>Wolbachia</ns4:italic> strains in diverse malaria vectors. Novel <ns4:italic>Wolbachia</ns4:italic> strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other <ns4:italic>Anopheles</ns4:italic> mosquito species, which could be used for population replacement or suppression control strategies. </ns4:p>